Cytokines trigger changes in gene expression by modifying the activity of otherwise latent transcription factors (Hill and Treisman, 1995). Nuclear factor .kappa.B (NF-.kappa.B) is a 1prominent example of how such an external stimulus is converted into an active transcription factor (Verma et al., 1995). The NF-.kappa.B system is composed of homo- and heterodimers of members of the Re1 family of related transcription factors that control the expression of numerous immune and inflammatory response genes as well as important viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994). The activity of NF-.kappa.B transcription factors is regulated by their subcellular localization (Verma et al., 1995). In most cell types, NF-.kappa.B is present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit. This heterodimer is sequestered in the cytoplasm in association with I.kappa.B.alpha. a member of the I.kappa.B family of inhibitory proteins (Finco and Baldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). I.kappa.B.alpha. masks the nuclear localization signal of NF-.kappa.B and thereby prevents NF-.kappa.B nuclear translocation. Conversion of NF-.kappa.B into an active transcription factor that translocates into the nucleus and binds to cognate DNA sequences requires the phosphorylation and subsequent ubiquitin-dependent degradation of I.kappa.B.alpha. in the 26s proteasome. Signal-induced phosphorylation of I.kappa.B.alpha. occurs at serines 32 and 36. Mutation of one or both of these serines renders I.kappa.B.alpha. resistant to ubiquitination and proteolytic degradation (Chen et al., 1995); DiDonato, 1996 #370, Roff, 1996 #397.
The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are among the physiological inducers of I.kappa.B phosphorylation and subsequent NF-.kappa.B activation (Osborn et al., 1989; Beg et al., 1993). Although TNF and IL-1 initiate signaling cascades leading to NF-.kappa.B activation via distinct families of cell-surface receptors (Smith et al., 1994; Dinarello, 1996), both pathways utilize members of the TNF receptor-associated factor (TRAF) family of adaptor proteins as signal transducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b). TRAF proteins were originally found to associate directly with the cytoplasmic domains of several members of the TNF receptor family including the 75 kDa TNF receptor (TNFR2), CD40, CD30, and the lymphotoxin-.beta. receptor (Rothe et al., 1994; Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al., 1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). In addition, TRAF proteins are recruited indirectly to the 55 kDa TNF receptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996). Activation of NF-.kappa.B by TNF requires TRAF2 (Rothe et al., 1995; Hsu et al., 1996). TRAF5 has also been implicated in NF-.kappa.B activation by members of the TNF receptor family (Nakano et al., 1996); Ishida, 1996 #240. In contrast, TRAF6 participates in NF-.kappa.B activation by IL-1 (Cao et al., 1996b). Upon IL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinase that binds to the IL-1 receptor complex (Cao et al., 1996a); Huang, 1997 #400.
The NF-.kappa.B-inducing kinase (NIK) is a member of the MAP kinase kinase kinase (MAP3K) family that was identified as a TRAF2-interacting protein (Malinin et al., 1997). NIK activates NF-.kappa.B when overexpressed, and kinase-inactive mutants of NIK comprising its TRAF2-interacting C-terminal domain (NIK.sub.(624-947)) or lacking two crucial lysine residues in its kinase domain (NIK.sub.(KK429-430AA)) behave as dominant-negative inhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-.kappa.B activation (Malinin et al., 1997). Recently, NIK was found to associate with additional members of the TRAF family, including TRAF5 and TRAF6. Catalytically inactive mutants of NIK also inhibited TRAF5- and TRAF6-induced NF-.kappa.B activation, thus providing a unifying concept for NIK as a common mediator in the NF-.kappa.B signaling cascades triggered by TNF and IL-1 downstream of TRAFs. Recently two NIK-interacting protein designated characterized as novel human kinase I.kappa.B Kinases, IKK-.alpha. and IKK.beta. have been reported (Woronicz et al., 1997; Mercurio et al. 1997; Maniatis, 1997). Catalytically inactive mutants of IKK suppress NF-.kappa.B activation induced by TNF and IL-1 stimulation as well as by TRAF and NIK overexpression; transiently expressed IKK associates with endogenous I.kappa.B.alpha. complex; and IKK phosphorylates I.kappa.B.alpha. on serines 32 and 36.
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